The invention concerns compounds useful in determing the amount of active human leucocyte elastase in a body fluid or other sample. Human leucocyte elastase (HLE) is a therapeutic target implicated in a number of chronic inflammatory dieseases characterized by extensive destruction of proteinaceous components of structural tissue. These include inflammatory conditions resulting in connective tissue destruction, e.g., rheumatoid arthritis, emphysema, bronchial inflammation, osteoarthritis, spondylitis, lupus, psoriasis atherosclerosis, sepsis, septicemia, shock, periodontitis, cystic fibrosis, acute respiratory distress syndrome, and reperfusion injury. High levels of active HLE are found in the sputa samples from patients with cystic fibrosis and chronic bronchitis (ref). Irreversible inhibitors of HLE are believed to be useful therapeutic agents and should decrease the concentration of active elastase in the biological fluids of patients. See (Davies, P., (1991) Annals of the New York Academy of Sciences, ed. Weinbaum Giles and Krell, vol 624, pp 219-229).
The concentration of elastase in biological samples can be determined by immunological techniques (ref) although this will not necessarily distinguish between active and inactive enzyme. Active HLE can be measured in terms of its ability to hydrolyse a given substrate. However, this technique could result in underestimating the concentration of active elastase due to the presence of endogenous substrates or reversible inhibitors. In particular, HLE found in human sputa samples may be tightly complexed to the reversible inhibitor bronchial antileucoprotease (ALP, Gauthier, F., Fryksmark, U., Ohlsson, K., and Bieth, J. G. (1982) Biochim. et Biophys. Acta 700, 178-183). Irreversible active site titrants that utilize the catalytic machinary of the enzyme, require active enzyme and would be ideal for this purpose. Ideal active site titrants react specifically, rapidly, and stoichiometrically with the target enzymes. In addition irreversible active site titrants should produce stable enzyme-inhibitor complexes. Prior active reagents that meet these criteria are radiometric (e.g., radiolabeled diisopropylfluorophosphate as disclosure by Cohen, J. A., Oosterbann, R. A.,. and Berends, F., Methods Enzymol. 11, 81). Unfortunately, this reagent not only reacts with serine proteases but serine esterases as well. Initial active site titrants of serine proteases were often nonspecific. For example, 4-nitrophenyl-4-guanidinobenzoate developed for trypsin-like proteases also reacts with chymotrypsin (Chase, T. and Shaw, E. (1970) Methods Enzymol. 19, 20-27). More recently Gupton et al. (1984) and Powers et al. have pioneered the use of aza-peptides as active site titrants of serine proteases (see Gupton, B. F., Carroll, D. L., Tuhy, P. M., Kam, C-M., and Powers, J. C. (1984) J. Biol. Chem. 259, 4279-4287; and Powers, J. C. and Carroll, D. L. (1975) Biochem. Biophys. Res. Commun. 67, 639). For a review of this class of inhibitors see Powers, J. C. and Gupta, B. F. Methods Enzymol. 46, 208-216. The specificity can be controlled by the peptide sequence. While they are more specific, a reagent such as Z-Ala-Ala-Pro-Aala-ONp originally developed for elastase also reacts with chymotrypsin and subtilisin (Powers and Carroll, 1975). In addition, these reagents may react with nonspecific esterases or be degraded by other proteases found in biological samples. These reagents also form relatively labile enzyme-inhibitor complexes with half-lives of approximately 1 hour at 25.degree. C. (ref). Aprotinin derivatives described by Mehlich et al. (1988) fare somewhat better in that they produce complexes with half-lives an order of magnitude more stable but the reagents are proteins (58 amino residues) and thus less practical to work with due to cost (Melich, A., Beckmann, J., Wenxel, H. R., and Tschesche, H. (1988) BBA 957, 420-429. Powers, J. C. Boone, R., Carroll, D. L., Gupton, B. F., Kam, C-M., Nishino, N., Sakamoto, M., and Tuhy, P. M. (1984) J. Biol. Chem. 4288-4294.)